Molecular and functional interaction between protocadherin-γC5 and GABAA receptors.

1Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269, USA.

Abstract

We have found that the γ2 subunit of the GABA(A) receptor (γ2-GABA(A)R) specifically interacts with protocadherin-γC5 (Pcdh-γC5) in the rat brain. The interaction occurs between the large intracellular loop of the γ2-GABA(A)R and the cytoplasmic domain of Pcdh-γC5. In brain extracts, Pcdh-γC5 coimmunoprecipitates with GABA(A)Rs. In cotransfected HEK293 cells, Pcdh-γC5 promotes the transfer of γ2-GABA(A)R to the cell surface. We have previously shown that, in cultured hippocampal neurons, endogenous Pcdh-γC5 forms clusters, some of which associate with GABAergic synapses. Overexpression of Pcdh-γC5 in hippocampal neurons increases the density of γ2-GABA(A)R clusters but has no significant effect on the number of GABAergic contacts that these neurons receive, indicating that Pcdh-γC5 is not synaptogenic. Deletion of the cytoplasmic domain of Pcdh-γC5 enhanced its surface expression but decreased the association with both γ2-GABA(A)R clusters and presynaptic GABAergic contacts. Cultured hippocampal neurons from the Pcdh-γ triple C-type isoform knock-out (TCKO) mouse (Pcdhg(tcko/tcko)) showed plenty of GABAergic synaptic contacts, although their density was reduced compared with sister cultures from wild-type and heterozygous mice. Knocking down Pcdh-γC5 expression with shRNA decreased γ2-GABA(A)R cluster density and GABAergic innervation. The results indicate that, although Pcdh-γC5 is not essential for GABAergic synapse formation or GABA(A)R clustering, (1) Pcdh-γC5 regulates the surface expression of GABA(A)Rs via cis-cytoplasmic interaction with γ2-GABA(A)R, and (2) Pcdh-γC5 plays a role in the stabilization and maintenance of some GABAergic synapses.

Diagram of the Pcdh-γC5 constructs and in vitro interaction of Pcdh-γC5 CD with γ2IL-GABAAR subunit

(A)cMycPcdh-γC5 (Full) has 6 cadherin repeats (C1–C6) in the ectodomain. An exon encodes the variable region (orange), which has an amino acid sequence specific for Pcdh-γC5. The distal part of the cytoplasmic domain (blue) is common to all Pcdh-γs and is encoded by three constant exons. The membrane bound cMycPcdh-γC5-extra (Extra) has the six cadherin repeats but the cytoplasmic domain is missing. The membrane bound cMycPcdh-γC5-intra (Intra) has the cytoplasmic domain and a small piece of the ectodomain but the six cadherin repeats are missing. GS113 was isolated by Y2H from a rat brain library using the γ2IL as bait. It corresponds to the cytoplasmic Pcdh-γC5. The arrows show the location of the cMyc epitope. The asterisk shows the position of the epitope (N-terminus) recognized by the Rb anti-Pcdh-γC5. The cMyc and N-terminus epitopes are present in the three cMycPcdh-γC5 membrane-bound constructs. The membrane bound Extra-EGFP has the whole cytoplasmic domain of Pcdh-γC5 replaced by the EGFP tag (green). The diagrams depict the mature membrane-bound Pcdh-γC5 constructs after removal of the signal peptide. (B) In vitro pull down of bacterial fusion proteins. The upper double immunofluorescence blot shows that His-Pcdh-γC5 CD (γC5, red asterisk) was pulled down by GST-γ2IL. However, His-Pcdh-γA3 CD (γA3), His-Pcdh-γC3 CD (γC3) or His-Pcdh-α4 CD (α4) was not pulled down by GST-γ2IL. All lanes show that GST-γ2IL (green asterisk) was eluted by glutathione. The lower immunoblot shows the input of the corresponding Pcdh CDs. (C) Double immunofluorescence blot. His-Pcdh-γC5 CD (red band in lane 1) was pulled down by GST-γ2IL (green band, lane 1) but not by GST (green band, lane 2) even when more GST was bound to the glutathione beads. His-Pcdh CD fusion proteins were detected with a Ms anti-His mAb while GST or γ2-GST were detected with a Rb anti-GST Ab. In each blot, the left lane has protein markers (red protein bands) with the molecular weight shown in kD (black numbers at the left).

(A) The anti-Pcdh-γC5 antiserum precipitates [3H]FNZ binding activity of solubilized GABAARs from a rat forebrain membrane fraction. The total [3H]FNZ binding activity precipitated by 50 µl of anti-Pcdh-γC5 antiserum (27.9±3.8%) is significantly higher (n=4, *** p<0.001 in two-tail Student’s t-test) than both the non-specific binding activity (not displaceable by excess of clonazepam) that was precipitated by this antiserum (4.9±0.5%) and the binding activity precipitated by 50µl of the preimmune serum (PIS) (4.9±0.6%) from the same rabbit that produced the anti-Pcdh-γC5 antiserum. Thus, the specific binding (total binding minus non-specific binding) is 23%. (B) Anti-γ2 and anti-α1 GABAAR subunit antisera (made in GP) but not the γ2 GP PIS precipitate the 120kD Pcdh-γC5 protein (asterisk and arrow), as shown by immunoblotting with Rb anti-Pcdh-γC5. In addition, the γ2 antiserum precipitates a stronger protein band of 140 kD (arrowhead). The α1 antiserum also precipitates the 140 kD protein to a lesser extent. The PIS in B-E corresponds to preimmune serum from the same rabbit that produced the anti-γ2 antiserum. (C) The immunoreactivity of anti-Pcdh-γC5 with the immunoprecipitated 120 kD and 140 kD proteins was blocked by 50 µg/ml of antigenic peptide. (D) Antibody Pcdh-γC5(C) to the C terminus, which recognizes all members of the Pcdh-γ family, reacts with both the 120 kD (arrow and asterisk) and 140 kD (arrowhead) proteins co-precipitated with γ2 and α1 antisera, but not with the γ2 PIS. (E) The anti-γ2 or anti-α1 precipitates were digested with Endo H or PNGase F followed by SDS-PAGE and immunoblotting with anti-Pcdh-γC5. Endo H did not affect the mobility of the 120kD (arrow and asterisks) or 140 kD (arrowhead) Pcdh-γC5 proteins, while PNGase F increased the mobility of both proteins. The 140 kD protein (arrowhead) was also present in the input, although to a much lower concentration than the 120 kD protein. The input image in the first two left lanes is the same, but the first lane was exposed for a longer time to better visualize the 140 kD protein band. The numbers at the left of the immunoblots show the position of the molecular weight protein markers in kD.

Pcdh-γC5 helps the γ2 GABAAR subunit translocate to the surface of HEK293 cells and form clusters that co-localize with Pcdh-γC5 clusters

(A–I) Immunofluorescence of HEK293 cells co-transfected with non-tagged γ2 and one of the cMycPcdh-γC5 constructs followed by live-cell incubation with a mixture of GP anti-γ2, Ms anti-cMyc and Rb anti-Pcdh-γC5. The γ2 subunit, when co-expressed with either cMycPcdh-γC5 (Full+γ2, A–C) or cMycPcdh-γC5-intra (Intra+γ2, D–F), formed surface γ2 clusters that frequently co-localized with those of cMycPcdh-γC5 (arrowheads, A–C) and cMycPcdh-γC5-intra (arrowheads, D–F). However, the γ2 subunit, when was co-expressed with cMycPcdh-γC5-extra (Extra+γ2, G–I), formed no γ2 clusters at the cell surface (G) while cMycPcdh-γC5-extra formed surface clusters (H). The labeling with anti-Pcdh-γC5 (not shown) was identical to that of anti-cMyc antibody. (J–L) HEK293 cells were transfected with the non-tagged γ2 subunit alone followed by live-cell incubation with GP anti-γ2 antibody followed by fixation, permeabilization and incubation with Rb anti-γ2 antibody. The γ2 subunit was expressed in HEK293 cells (K) but not on the surface of the same cells (J). The nuclear DAPI staining is shown in the overlay (L). Scale bar = 5 µm. (M) Immunoblots of HEK293 cells, non-transfected (NT), or transfected with EGFP-γ2 only (γ2) , or co-transfected with EGFP-γ2 and one of the cMycPcdh-γC5 constructs (γ2+Full; γ2+Extra; γ2+Intra). Cell surface proteins were biotinylated, affinity-purified on Neutravidin-agarose, subjected to SDS-PAGE and immunoblotting with Rb anti-EGFP antibody (upper blot). The 70 kD EGFP-γ2 protein band is indicated by an arrow. The actin loading control (total cell lysate) shows that the amount of protein in the cultures, used for the purification of the biotinylated proteins was similar under the various transfection conditions. The lower blots are two regions of the same immunoblot, with anti- Pcdh-γC5, of transfected HEK293 total cell lysates showing the expression of the three cMycPcdh-γC5 constructs of the expected mobility: Full (120 kD), Extra (98 kD) and Intra (32 kD). The protein bands of the three expressed constructs are indicated by a black dot on the left side of the protein band. The graph on the right side shows the quantification of the EGFP-γ2 protein band of the upper immunoblot blot normalized for actin. (n = 2 experiments, * p<0.05; ** p<0.01 in one-way ANOVA Tukey-Kramer multiple comparison test).

In co-transfected HEK293 cells, Pcdh-γC5 induces the formation of large GABAAR clusters at the cell surface, many of which co-localize with Pcdh-γC5 clusters

(A–C) Immunofluorescence of HEK293 cells co-transfected with non-tagged GABAARs α1, β3 and γ2 subunits and incubated with γ2, β3 and α1 antibodies under live-cell conditions. The three antibodies GP anti-γ2 (A), Ms anti-β2/3 (B) and Rb anti-α1 (not shown) subunits showed identical immunofluorescence pattern characterized by the presence of microclusters on the surface of HEK293 cells. (D–L) HEK293 cells co-transfected with the three GABAAR subunits and one of the cMycPcdh-γC5 constructs were incubated with GP anti-γ2 (red, D,G, J), Ms anti-cMyc (green, E,H,K) and Rb anti-Pcdh-γC5 (not shown) under live-cell incubation condition. Co-transfection of GABAARs with cMycPcdh-γC5 (Full+α1β3γ2, D–F) and cMycPcdh-γC5-intra (Intra+α1β3γ2 , G–I) induced the formation of large γ2 clusters on HEK293 cell surface, many of them colocalizing with the Pcdh-γC5 clusters. Arrowheads point to colocalization of γ2 and cMycPcdh-γC5 construct clusters. In contrast, co-transfection of HEK293 cells with GABAARs and cMycPcdh-γC5-extra (Extra+α1β3γ2, J–L) shows few γ2 clusters on cell surface and only few cMycPcdh-γC5-extra clusters co-localize with γ2 clusters (arrowheads). In all of the experiments the labeling with anti-Pcdh-γC5 (not shown) was identical to that of anti-cMyc. Scale bar = 5 µm. (M) Quantification of the colocalization between γ2 and cMycPcdh-γC5 construct clusters (n=10 transfected cells per construct from 3 experiments, *** P<0.001, one-way ANOVA Tukey-Kramer multiple comparison test). (N) Immunoblots of HEK293 cells, non-transfected (NT) or co-transfected with α1, β3 and EGFP-γ2 (α1β3γ2), or co-transfected with cMycPcdh-γC5, α1, β3 and EGFP-γ2 (Full+α1β3γ2) after purification of biotinylated surface proteins. Immunoblot with Rb anti-EGFP antibody (upper blot) shows the 70 kD EGFP-γ2 protein band (arrow). Underneath are the total lysate loading actin control and the immunoblot with anti-Pcdh-γC5, which shows the presence of the 120kD cMycPcdh-γC5 (Full) protein band (marked by a dot on the left side) in the total lysates of cells co-transfected with Full+α1β3γ2, but not in the non-transfected cells or cell transfected with α1β3γ2.

Triple-label immunofluorescence of transfected HP neurons. (A, A1–A3, B1–B3) Neurons were transfected with cMycPcdh-γC5 and EGFP. (C, C1–C3, D1–D3)) Neurons were transfected with cMycPcdh-γC5-intra and EGFP. (E, E1–E3, F1–F3) Neurons were transfected with cMycPcdh-γC5-extra and EGFP. The transfected neurons show both green fluorescence and surface cMyc labeling (red). A1–A3, C1–C3 and E1–E3 show the fluorescence corresponding to the boxed area in A, C and E respectively. B1–B3, D1–D3 and F1–F3 are from sister cultures transfected with cMycPcdh-γC5, cMycPcdh-γC5-intra and cMycPcdh-γC5-extra respectively. The cultures were incubated with Ms anti-cMyc (red in all panels) under live-cell condition followed by fixation, permeabilization and incubation with sheep anti-GAD (blue in A, A1, C, C1, E and E1) or GP anti-γ2 (blue in B1, D1 and F1). Arrowheads show surface clusters of the cMycPcdh-γC5 constructs (red) that co-localize with GAD+ boutons (blue) or γ2-GABAAR clusters (blue). Panel C shows a transfected and a non-transfected neuron next to each other. The arrows point to GAD+ boutons contacting dendrites of the non-transfected neuron. In panel E the crossed arrow shows the growth cone of a GAD-containing axon. Scale bar is 5 µm. (G) Quantification of the density of the surface cMyc clusters on HP neurons transfected with the cMycPcdh-γC5 constructs (n = 21 neurons per construct from 7 transfection experiments). (H) Quantification of the density of γ2-GABAAR clusters on HP neurons transfected with the cMycPcdh-γC5 constructs (n = 40 or 45 neurons per construct from 8 or 9 transfection experiments) (I) Quantification of the percentage of the γ2 clusters that co-localize with clusters of the cMycPcdh-γC5 constructs (n = 25 or 26 neurons per construct from 5 transfection experiments). (J) Quantification of the number of GAD+ boutons contacting neurons transfected with the cMycPcdh-γC5 constructs (n = 15–21 neurons per construct from 6 transfection experiments). (K) Quantification of the percentage of GAD+ boutons co-localized with clusters of the cMycPcdh-γC5 constructs (n = 20 neurons per construct from 5 transfection experiments). For panels G–K: *** p<0.001; * p<0.001, one-way ANOVA Tukey-Kramer multiple comparison test.

(A–C) Neuronal cultures from the homozygous TCKO mouse. These neurons show abundant GABAergic synapses (A), lack of Pcdh-γC5 expression (B) and normal and abundant neuronal processes as shown by the anti-TUJ1 antibody (C). (D–F) Neuronal cultures from sister wild type mice. They show GABAergic synapses (D), expression of Pcdh-γC5 (E) and abundant neuronal processes (F). The boxed areas in A and D are shown at higher magnification in the smaller panels. Presynaptic VGAT boutons (blue) are apposed to gephyrin (red) and γ2-GABAAR clusters (green) as indicated by arrowheads. Gephyrin and γ2-GABAAR clusters co-localize with each other. Scale bar is 10 µm for all panels except for the enlarged boxed area, which correspond to 5.5µm. (G) Quantification of the density of GABAergic marker puncta in the WT and HMZ mice (n = 14 neurons per genotype from five coverslips). (H) Quantification of the % of GAD+ neurons in the WT and HMZ cultures (n=5 and 4 coverslips respectively). (I) Quantification of the % of TUNEL+ neurons in the WT and HMZ cultures (n = 4 and 3 coverslips respectively). Panels G–I: *** p<0.001; * p<0.001, two-tail Student t test).

Knocking down Pcdh-γC5 in hippocampal neurons leads to a decrease in both γ2-GABAAR cluster density and GABAergic innervation

(A and B) The sh1 and sh1 3m Pcdh-γC5 shRNAs used in this study. The sh1 3m carries three point mutations shown in red. (C–F) HP neurons co-transfected with sh1 and mCherry (C and E) have lower density of Pcdh-γC5 clusters (green), γ2-GABAAR clusters (blue in C) and GAD+ boutons (blue in E) than those co-transfected with sh1 3m and mCherry (D and F respectively). Right-side panels show enlargement of the boxed areas in the left panels. Scale bar = 9 µm for the left-side panels and 5 µm for the right side panels. (G–I) Quantification of the effect of knocking down Pcdh-γC5 (sh1 and mCherry) on the density of Pcdh-γC5 clusters, γ2-GABAARclusters and GAD+ boutons compared to various controls including a rescue mRNA. (n = 15 neurons per condition from 3 experiments, *** p<0.001; ** p<0.01, one-way ANOVA Tukey-Kramer multiple comparison test). (J) Immunoblots with antibodies to Pcdh-γC5 and actin of HEK293 co-transfected cells show that sh1 knocks down the protein expression of co-transfected cMycPcdh-γC5, however sh1 3m or the mU6 plasmid have no effect. (K) Quantification of the cMycPcdh-γC5 protein band intensities in the immunoblots of panel J (n = 2 experiments, *** p<0.01 in one-way ANOVA Tukey-Kramer multiple comparison test).